Technical Field
The present invention generally relates to molecular imaging compositions, and methods of using the same, and methods of signal amplification. More particularly, the invention relates to multiplexed and programmable imaging compositions for high resolution analysis of cell physiology, phenotype, and molecular pathway visualization.
Description of the Related Art
The chemical amplification of molecular recognition events is critical to numerous in situ analyses of proteins, RNA and other biomolecular species within cells and tissues. Such capabilities are often necessary to extend the dynamic range of an imaging technique so that dilute molecular targets can be visualized within a specimen. Several enzymatic signal amplification strategies have been established that allow large numbers of active dye molecules to be localized to their primary target including tyramide signal amplification (TSA) and polymerization chain reaction-based methods such as rolling circle amplification (RCA).
Non-enzymatic amplification procedures based on the triggered polymerization of nucleic acid hairpin devices have also been developed explicitly for the in situ detection of mRNA targets. Each of these methods offer high signal amplification gains and can be used to detect low level molecular species. However, they generally offer limited control over final amplification levels since they rely on chemical reactions that need to be timed and/or quenched in order to arrest the signal amplification process. Moreover, the rates of these chemical reactions likely depend on the local concentrations and chemical environment surrounding their primary targets, which can, in turn, lead to sample- and local context-dependent modulation of signal amplification rates and levels in different settings. In addition to increased detection sensitivities, a variety of molecular analyses stand to benefit from the development of convergent amplification strategies that can produce defined and uniform amplification gains that can be tuned predicatively to regulate reporting levels.
Such capabilities are important for comparative analyses of target levels within and across different biological samples since potential variability in protein staining can compromise abilities to assess functionally significant changes in target levels. Furthermore, the intensities of the reporting molecules must often be regulated for multiplexed molecular imaging strategies where multiple types of fluorescent reporting molecules are used to detect different molecular targets within a sample. The emission spectra of most fluorophores are relatively broad and exhibit a significant degree of spectral overlap. Since these properties can lead to appreciable bleed-through of target signals between a microscope's spectral channels, the levels of multiple fluorophores in multi-color imaging assays must often be balanced appropriately so as to avoid intense staining of one particular range of a hyperspectral imaging system and to ensure the detection of dilute targets is not influenced by noise generated by spatially and spectrally overlapping signals stemming from more abundant targets within a sample.
These issues are typically addressed by diluting the recognition reagents of the more intense targets within a sample to achieve more equitable intensity distributions among each overlapping target. Although it would clearly be beneficial to balance maker intensities by amplifying the less intense signals, the lack of control over amplification levels provided by existing technologies generally limits their use in these applications.